Honours Projects - Dr Thomas Fath

Background

The actin microfilaments are composed of a two stranded polymer of actin and most filaments also contain polymers of the rod shaped tropomyosin running along the major groove in the filament.

As you heard in the lecture on Microfilaments, the different tropomyosin isoforms have the ability to direct the assembly of specific cellular structures.

Previous experiments have been performed in which the neuroblastoma derived cell line, B35 (Schubert D, etal., 1974) is given a gene construct driving the expression of a specific tropomyosin. Cells are then isolated which express the introduced tropomyosin and analysed for changes in phenotype.

Thus far we have shown that:

Tm5NM1/2 drives the formation of stress fibres and results in cells with arced membranes and completely lacking lamellipodia.

TmBr3 drives the formation of very broad lamella.

Tm3 drives the formation of filipodia.

We have now introduced a new tropomyosin into B35 cells, Tm4.

Description of B35 Phenotype Classifications

During today's laboratory it may be useful to download the above image and open on the desktop for comparison beside the images you are analysing.

Neurite Definition - a projection which is narrower than the smallest diameter of the nucleus for at least half it’s length and is greater in length than the widest diameter of the nucleus.

WT Undifferentiated B35 cells can be described as having the following types of phenotypes.

1. "Fan" phenotype

such that a single continuous lamellum extends distally in all directions from the soma to encircle the nucleus and soma entirely.

2. "Broken fan" phenotype

As “Fan” phenotype, except that a single lamellum extends from the soma distally to encircle between 30% and 100% of the soma perimeter.

The cell may or may not extend a single neurite, not longer than 3x the widest diameter of the nucleus.

3. "Stumped" phenotype

Cell soma has multiple (i.e. two or more) stalks of lamella extending distally from the soma.

The longest distance between the most distal edge of lamella (i.e. the lamellipodia) and the nucleus is not more than 3x the width of the nucleus at its widest point.

In addition, soma may extend neurites, but not greater in length than 3x the widest diameter of the nucleus.

4. "Pronged" phenotype

The cell extends at least one neurite, and no one neurite is longer than 3x the width of the nucleus at its widest diameter.

A single (but not multiple) lamellum encircling less than 30% of the soma perimeter may or may not extend from the soma.

5. "Stringed" phenotype

At least one neurite (as defined above) extends distally from the soma.

And at least one neurite is a length exceeding that of 3x the diameter of the nucleus at its widest part.

Cell may or may not extend lamella in addition to this.

6. “Pygnotic” phenotype

Cells comprising a nucleus, and an immediate lamellal fan around the nucleus.

lamellal fan does not extend distally at any point more than the length of the narrowest diameter of the nucleus.

Analysis Notes

Where neurites or lamella from 2 or more cells are overlapping and you can't distinguish them, exclude these cells from the analysis. If you feel confident to discern the morphology of both cells, you can go ahead with including them in the analysis. Important is, that you are consistent in doing this between the two experimental groups.

Cells which are in any part occluded by the perimeter of the field of view will not be included, unless that cell is a stringed cell, and the part being occluded is the neurite which has at least a length of 3x the width of the nucleus at its widest part within the field of view.

When two nuclei are less than the distance of diameter of one nuclei apart, they will be considered as undergoing mitosis, and counted as one cell.